Circular RNA-VPS13A attenuates diabetes-induced enteric glia damage by targeting miR-182/GDNF axis

Gastrointestinal (GI) complications of diabetes mellitus (DM) significantly impact on patients’ quality of life. Enteric glial cells (EGC) are the key cell type of enteric nervous system (ENS), which contributes to the destruction of gut homeostasis in DM. Circular RNAs (circRNAs) are a novel type of RNAs abundant in the eukaryotic transcriptome, which form covalently closed continuous loops. In this study, the contribution of circRNAs to EGC damage in DM is investigated. Transcriptome sequencing analysis and functional study show that circVPS13A is significantly down-regulated in hyperglycemia-treated EGC, and circVPS13A overexpression attenuates EGC damage in both in vitro and in vivo DM models. In vitro mechanistic study using dual-luciferase reporter assay, affinity-isolation assay, fluorescence in situ hybridization (FISH) and immunostaining analysis identify that circVPS13A exerts its protective effect by sponging miR-182 and then up-regulates glial cell line-derived neurotrophic factor (GDNF) expression. In addition, in vivo study confirms that the circVPS13A-miR-182-GDNF network regulation can attenuate hyperglycemia-induced EGC damage of duodenum in streptozotocine (STZ)-induced DM mice. The findings of this study may provide novel insights into the protective role of circVPS13A in DM-associated EGC damage and clues for the development of new therapeutic approaches for the prevention of GI complications of DM.


Introduction
Diabetes mellitus (DM) widely influences organs in the body, and gastrointestinal (GI) complications are more common in diabetic patients than in healthy subjects [1][2][3]. Thus, better managing GI complications is important for the control of DM and the improvement of patients' quality of life. The pathogenesis of GI complications in DM is complex. During its pathogenic process, dysfunction of enteric nervous system (ENS) is a leading cause with great clinical importance [4,5]. Enteric glial cells (EGC) are the main type of cells in ENS, which form an extensive network in GI tract to protect enteric neurons [6,7]. In a previous study, changes of the morphology and number of EGC were observed in the gastric motility dysfunction in DM [8]. In addition, it has been postulated that in the acute diabetes, EGC are activated to protect enteric neurons from damage via promoting neurotrophin release [9]. Therefore, enteric glia is responsible for gut motility reflexes, barrier function and inflammation in diabetes-associated GI complications. Understanding the mechanism underpinning enteric glia dysfunction in such pathogenetic process may contribute to exploring novel therapeutic approaches to better control GI complications associated with DM. Circular RNAs (circRNAs) are a new class of noncoding RNAs, which are likely related to the occurrence and development of various human diseases [10,11]. Thus, elucidating the functional role of circRNAs may contribute to the understanding of disease pathogenesis [12,13]. Different types of non-coding RNAs including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) have been shown to be key regulators in the development of DM [14,15]. However, little is known about the newly discovered circRNAs in the pathological process of DM. Lisa et al. [16] revealed that cir-cRNAs are novel regulators of β-cell activities and suggested their involvement in β-cell dysfunction in DM. Caspase-1-associated circRNA (CACR) likely leads to diabetic cardiomyopathy (DCM) pyroptosis via the miR-214-3p/caspase-1 pathway [17]. Induction of cZNF532 regulates pericyte biology by acting as a miR-29a-3p sponge and inducing the expressions of NG2, LOXL2, and CDK2, which is an exploitable therapeutic approach for the treatment of diabetic retinopathy [18]. Up till now, it still remains unclear about the role of circRNAs in diabetes-associated GI complications.
In this study, we found that circVPS13A is significantly downregulated in hyperglycemia-treated EGC based on transcriptome sequencing data, and further investigated the role of circVPS13A in the diabetes-induced enteric glia damage. This study may lead to a new therapeutic approach for the treatment of diabetes-associated GI complications.

Materials and Methods
Cell culture and transfection

TUNEL assay
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay Kit (Beyotime, Nantong, China) was used to detect apoptotic cells. Cells or tissues were fixed with 4% paraformaldehyde for 30 min and treated with 0.5% Triton X-100-PBS for 5 min according to the manufacturer′s instructions. Following this, the sections were then incubated with a terminal deoxynucleotidyl transferase reaction mix for 60 min at 37°C, followed by DAPI staining for 5 min. TUNEL-positive cells (red staining cells) were observed via fluorescence microscopy (Olympus, Tokyo, Japan).

STZ-induced diabetic mouse
C57BL/6J mice were purchased from Changzhou Cavens Experimental Animal Center (Changzhou, China). DM was induced by intraperitoneal injection of STZ (Sigma). Mice received STZ (50 mg/kg in 10 mM citrate buffer, pH 4.5) injection for 5 consecutive days. Control mice were administered with the same volume of sodium citrate buffer. All mice were kept in an airconditioned room with a 12-hour light-dark cycle and fed with standard laboratory chow, with free access to water. Seven days after the final STZ injection, blood samples were collected from the tail vein for the measurement of blood glucose level. The mice with a blood glucose level of over 300 mg/dL were considered as DM. All animal experiments were performed in accordance with the institutional guidelines of Jiangsu Institute of Nuclear Medicine (Wuxi, China) for the Care and Use of Laboratory Animals, and this study was approved by the Ethics Committee of Jiangsu Institute of Nuclear Medicine.

Transcriptome sequencing
CRL-2690 cells were incubated with 25 mM glucose or 300 mM glucose for 24 h. After incubation, RNA samples were prepared from cells for transcriptome sequencing by Novogene (Beijing, China) . The clustering of samples was processed through the cBot Cluster Generation System (Illumia, San Diego, USA) according to the manufacturer's instructions. After cluster generation, the library was sequenced on the Illumina Hiseq platform and 125 bp/150 bp pairedend reads were generated. HTSeq v0.6.0 was used to count the number of reads mapped to each gene. FPKM of each gene was calculated based on the gene length with reads count mapped individually.

Western blot analysis
Cells were treated with RIPA lysis buffer for 15 min prior to total protein extraction. Protein concentration was determined by BCA assay. Samples (20 μg) were subject to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, USA). The membrane was blocked with 5% bovine serum albumin (BSA) for 1 h and then incubated with the anti-GDNF antibody (1: 1000; Abcam, Cambridge, USA) or anti-GAPDH antibody (1:1000; Abcam), followed by incubation with the corresponding HRP-conjugated secondary antibody (1:500 ; Beyotime). The protein bands were visualized using an ECL assay kit (Beyotime) and quantified using ImageJ (National Institutes of Health, Bethesda, USA). GAPDH was used as the loading control.

Enzyme-linked immunosorbent assay (ELISA)
After treatment, the culture medium was collected and processed for ELISA. Culture medium (100 μL) was reacted with the GDNF ELISA kit (Solarbio, Beijing, China) according to the manufacturer′s instructions. The absorbance was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, USA).

RNA pull-down assay
CRL-2690 cells were transfected with 3′-end biotinylated miR-182 (Genepharma) at a final concentration of 50 nM for 24 h. Then, the cells were lysed and centrifuged as described previously [19]. A total of 50 μL aliquots of the samples were prepared as input. The remaining lysates were incubated with M-280 streptavidin magnetic beads (Invitrogen, Carlsbad, USA) for 2.5 h at 4°C. Then the beads were washed twice with ice-cold lysis buffer, twice with low-salt buffer, and once with high-salt buffer. The bound RNAs were isolated using TRIzol reagent for the measurement of circVPS13A by qPCR.

Fluorescence in situ hybridization (FISH) analysis
RNA FISH experiments were carried out using the kit purchased from Genepharma according to the manufacturer′s instructions. The probes of circVPS13A and miR-182 (labeled with Cy3 dye) were as follows: circVPS13A, CAAGAATACCATGTATAGCATAAGCCCACT TTAGGGCTTGTGCTCCTGGTCTTTGCAC; miR-182, CGGTGTGAG TTCTACCATTGCCAAA. All hybridizations were done overnight in the dark at 37°C in a humidified chamber. For protein FISH experiments in cells, CRL-2690 cells were cultured on coverslips, fixed with 4% paraformaldehyde for 20 min and incubated in PBS overnight at 4°C, followed by the detection of circVPS13A and miR-182 as previously reported [19]. The coverslips were heated to 65°C for 5 min in hybridization buffer containing digoxigenin-labeled miR-182 or biotin-labeled circVPS13A probe, and then hybridized overnight at 37°C. The coverslips were washed and blocked for 1 h at room temperature. After that, coverslips were incubated with a Cy3conjugated anti-digoxigenin antibody overnight at 4°C and then cultured with FITC-streptavidin for 2 h at 4°C. The cell nuclei were stained with DAPI. Finally, slides were examined under a fluorescence microscope (Olympus) and digital images were captured.

Digital PCR analysis
The chip-based digital PCR (dPCR) was conducted on a Quant-Studio 3D Digital PCR System (Life Technologies) for the absolute quantification of the copy number of circVPS13A and miR-182. cDNA was synthesized from RNA isolated from EGC under indicated conditions, and digital PCR was conducted using the following reaction conditions: hot start at 96°C for 10 min, denaturation at 98°C for 30 s, annealing/extension at 62°C for 2 min for a total of 39 cycles, followed by a final extension step at 65°C for 2 min. The data analysis was performed with QuantStudio 3D AnalysisSuite Cloud Software version 3.1.2. The primers used are: circVPS13A F, 5′-TGAAATTCTTGCAGAAATGTTG-3′, R, 5′-GTGCTCCTGGTCTTTGCACAAT-3′; miR-182 F, 5′-ATCACTTTTGG CAATGGTAGAACT-3′, R, 5′-TATGGTTTTGACGACTGTGTGAT-3′.

Immunofluorescence analysis
The section of mouse duodenum was cut into 35-μm slices with a cryostat. Then, the sections were incubated with anti-GFAP antibody (Sigma) overnight at 4°C. On the following day, the sections were washed and incubated with Alexa Fluor 488-conjugated antimouse IgG for 1 h. After wash with PBS, the sections were incubated with ProLong gold Antifade reagent containing DAPI (Invitrogen) for visualization of the nuclei. Immunofluorescence images were captured with an Olympus DP73 fluorescence microscope (Olympus).

Intestinal intramuscular injection
Virus injections were performed at 2 weeks after the establishment of DM mice model. The mice were divided into three groups: DM group, DM+circVPS13A OV (Overexpression), DM+circVPS13A OV+miR-182 TF (Transfection). Mice were anesthetized using isoflurane (2%-4%) and kept at a constant body temperature. For the experiments, circVPS13A was subcloned into pHBAAV-CMV-circRNA-EF1-ZsGreen vector, and the vector or micrON™ miR-182 agomir was packaged by AAV9, then the injection of 2 μL AAV9-circVPS13A (1.2×10 11 GC/ml) or AAV9-miR-182 agomir (1×10 11 GC/ml) was conducted using a 10-μL Hamilton syringe into the wall of the duodenum at two sites near the myenteric plexus as previously reported [20]. Two weeks after intestinal intramuscular injection, the efficacy of AAV9 delivery was assessed by evaluating the expressions of circVPS13A or miR-182 in duodenum by qPCR and RNA FISH.

Statistical analysis
SPSS19.0 software was used to analyze data. Data were expressed as the mean±SD. Statistical comparisons were made by Student's ttest between two groups and one-way ANOVA followed by Tukey's post hoc test among different groups. P<0.05 indicates statistically significant difference.

circVPS13A expression is significantly down-regulated in both in vitro and in vivo DM models
To identify hyperglycemia-regulated circRNAs, CRL-2690 cells were cultured with 25 mM glucose (normal glucose, NG) or 300 mM glucose (high glucose, HG) for 24 h. First, cell viability was assessed by TUNEL assay and the results showed that HG significantly reduced cell viability in CRL-2690 cells ( Figure 1A). The RNA samples of NG and HG groups were prepared for transcriptome sequencing, and 4317 differentially expressed circRNAs were identified between the HG group and NG group, with 3797 upregulated and 520 downregulated ( Figure 1B). Among these altered circRNAs, circ_0003641 was found to be the most down-regulated circRNA, which has a homologous gene named VPS13A in rat genome with a sequence similarity of over 85% ( Figure 1C). circVPS13A was found to be resistant to RNase R digestion, whereas linear VPS13A mRNA responded to RNase R readily ( Figure 1D). Whether hyperglycemia influences circVPS13A expression in vitro and in vivo was further evaluated. qRT-PCR revealed that HG reduced circVPS13A expression in CRL-2690 cells after 24 h of treatment ( Figure 1E). Meanwhile, reduced circVPS13A was observed in the duodenum and EGC of diabetic mice at 6 weeks post STZ-induction ( Figure 1F,G). These data indicated that hyperglycemia significantly down-regulated circVPS13A expression in both in vitro and in vivo DM models.

circVPS13A overexpression attenuates EGC damage in both in vitro and in vivo DM models
To understand the function of circVPS13A under hyperglycemia, 1001 VPS13A attenuates glia damage by miR-182/GDNF circVPS13A was up-regulated by circVPS13A overexpression in both in vitro and in vivo DM models ( Figure 2A). First, circVPS13A overexpression reduced the cytotoxicity and apoptosis of CRL-2690 cells under hyperglycemia ( Figure 2B,C). In addition, the up-regulated expression of glia marker GFAP was observed, indicating that circVPS13A attenuated EGC damage under hyperglycemia ( Figure  2B,C). Then, the inhibitory effect of circVPS13A overexpression on EGC damage in STZ-induced diabetic mice was evaluated by intestinal intramuscular injection of AAV packaged circVPS13A expression plasmid. The injection up-regulated the expression of circVPS13A in EGC of duodenum, and subsequently reduced apoptosis of EGC and increased EGC density in vivo ( Figure 2D,E). These data indicated that hyperglycemia-induced EGC damage could be attenuated by circVPS13A overexpression in both in vitro and in vivo DM models.

circVPS13A attenuates EGC damage by acting as a sponge for miR-182
To understand the mechanism of the regulatory role of circVPS13A under hyperglycemia, the target genes of circVPS13A were analyzed using bioinformatics tools and dual-luciferase reporter assay. First, bioinformatics data predicted that circVPS13A exerts its function at the post-transcriptional level by acting as a miR-182 sponge ( Figure  3A). Then, dual-luciferase reporter assay identified that transfection with miR-182 mimic decreased the luciferase activity of LUC-cir-cVPS13A without influencing the luciferase activity of LUC-cir-cVPS13A mutant ( Figure 3B). Finally, RNA pull-down assay showed that circVPS13A was enriched in the miR-182-captured fraction ( Figure 3C). FISH assay revealed the co-expression of circVPS13A and miR-182 in the cytoplasm of CRL-2690 cells ( Figure 3D), and digital PCR showed the copies of circVPS13A were significantly down-regulated and the copies of miR-182 were slightly up-regulated in hyperglycemia-treated EGC compared to controls ( Figure 3E). In addition, miR-182 overexpression reversed the protective effect of circVPS13A overexpression on hyperglycemia-induced EGC damage ( Figure 4). These data indicated that circVPS13A attenuated hyperglycemia-induced EGC damage by acting as a sponge for miR-182

circVPS13A-miR-182-GDNF network is involved in regulating EGC damage in both in vitro and in vivo DM models
Targetscan database was used to predict the target genes of miR-182. GDNF is one of the candidate genes to be selected for further evaluation due to its role in glia activation. Luciferase reporter assay verified the direct regulation of miR-182 in GDNF expression; while miR-182 mimic significantly reduced GDNF expression at the protein level ( Figure 5A-C). In addition, GDNF expression was downregulated in hyperglycemia-treated EGC, and negatively correlated with miR-182 but positively correlated with circVPS13A ( Figure 5D, E). Further study also revealed that miR-182 inhibitor or cir-cVPS13A overexpression significantly enhanced GDNF expression at the protein level in hyperglycemia-treated EGC ( Figure 5D).

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VPS13A attenuates glia damage by miR-182/GDNF Meanwhile, GDNF silencing attenuated the protective effect of miR-182 inhibitor or circVPS13A overexpression on hyperglycemia-induced EGC cytotoxicity and apoptosis in CRL-2690 cells (Figure 6), indicating that circVPS13A overexpression acts similarly to that of miR-182 inhibitor. Finally, the role of the circVPS13A-miR-182-GDNF network in EGC damage was investigated in diabetic mice. circVPS13A overexpression led to the increased expression of GDNF, which was attenuated by transfection with miR-182 agomir ( Figure 7). These data indicated that the circVPS13A-miR-182-GDNF network regulation could attenuate hyperglycemia-induced EGC damage in both in vitro and in vivo DM models.

Discussion
Impaired function of the GI tract is the common complication of DM, and EGC dysfunction contributes to the destruction of gut homeostasis under pathophysiological conditions [21,22]. Therefore, it is urgent to develop new therapeutic strategies to control diabetes-related GI complications. In this study, we demonstrated that circVPS13A was significantly down-regulated in CRL-2690 cells under hyperglycemia based on transcriptome sequencing and qPCR, and that circVPS13A overexpression attenuated EGC damage in both in vitro and in vivo DM models. Furthermore, circVPS13A acted as a miR-182 sponge to sequester and inhibit miR-182 activity, which led to increased expression of GDNF. This study might provide novel insights into the understanding of the pathogenesis of DM-related GI complications. circRNAs exert their functions by acting as the sponges for miR-NAs or proteins, competing with the linear mRNA, or regulating the

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VPS13A attenuates glia damage by miR-182/GDNF transcription of their parental genes [23,24]. circRNAs are critically involved in metabolic processes of gestational diabetes mellitus (GDM) and related to insulin resistance and β-cell dysfunction [25,26]. VPS13A was previously identified as a key factor in various human neurodegenerative diseases [27,28]. And up to now, no literature showed the crypt expression of VPS13A in the small intestine and colon. Therefore, little is known about the function of circular RNA-VPS13A in the gut. In this study, we revealed that increased circVPS13A could sponge and sequester miR-182 in response to hyperglycemia, relieving its miR-182-mediated suppressive effect on EGC damage. MiR-182 plays a significant role in regulating neuronal axon outgrowth and dendrite tree formation [29]. Roser et al. [30] demonstrated that inhibition of miR-182 in GDNF-treated polymorphonuclear neutrophils (PMN) cultures diminished the beneficial effect of GDNF, suggesting that miR-182 is involved in mediating the effects of GDNF. Here, we found that  1005 VPS13A attenuates glia damage by miR-182/GDNF hyperglycemia enhanced the expression of miR-182, and that inhibition of miR-182 attenuated hyperglycemia-induced EGC damage both in vitro and in vivo, which is comparable to that of circVPS13A overexpression.
To further investigate the effects of miR-182, targetscan database and luciferase reporter assay were performed to confirm whether GDNF, an important factor in glia activation, is the direct target of miR-182. GDNF is known to participate in the regulation of EGC function, and altered GDNF expression leads to a higher susceptibility towards apoptosis, resulting in the disruption of the mucosal integrity and severity of inflammation [31]. Zhang et al. [32] reported that EGC can regulate intestinal epithelial barrier integrity indirectly via manipulating the release of GDNF in vivo [32]. Bau-man et al. [33] showed that EGC lost their barrier-enhancing function under morphine treatment due to the decreased production of GDNF [33]. In the current study, we also found that GDNF was significantly down-regulated in hyperglycemia-treated EGC; while circVPS13A overexpression or miR-182 inhibition significantly enhanced GDNF expression. Consistently, GDNF silencing attenuated the protective effect of circVPS13A overexpression or miR-182 inhibition on hyperglycemia-induced EGC cytotoxicity and apoptosis in CRL-2690 cells. In addition, our in vivo study confirmed the protective effect of circVPS13A overexpression on EGC damage. However, future studies are required on the GNDF knockdown mouse model to further investigate the molecular mechanism of circVPS13A/miR-182. In addition, besides modulating circVPS13A-

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VPS13A attenuates glia damage by miR-182/GDNF miR-182 signaling to enhance GDNF in EGC, normalizing GFAP+ enteric glial cells could also recover GDNF expression indirectly in duodenum, since enteric glial cells are a source of GDNF.
In conclusion, in this study we revealed that circVPS13A is an important regulator of EGC damage in DM. circVPS13A overexpression increases cell viability and inhibits apoptosis of EGC by sponging miR-182 and inducing GDNF release (Figure 8). Therefore, targeting the circVPS13A-miR-182-GDNF network may be a promising strategy for the treatment of DM-related GI complications.

Funding
This work was supported by the grants from the Project of Wuxi Health Commission (Nos. M202011 and Z202110).